Format

Send to

Choose Destination
Am J Physiol Lung Cell Mol Physiol. 2015 Jan 1;308(1):L33-47. doi: 10.1152/ajplung.00217.2014. Epub 2014 Oct 24.

A non-BRICHOS SFTPC mutant (SP-CI73T) linked to interstitial lung disease promotes a late block in macroautophagy disrupting cellular proteostasis and mitophagy.

Author information

1
Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania;
2
Department of Pediatrics; Monroe Carell Jr. Children's Hospital, Vanderbilt University, Nashville, Tennessee;
3
Department of Pediatrics; University of Colorado School of Medicine, Denver, Colorado;
4
Department of Pathology and Lab Medicine; University of North Carolina, Chapel Hill, North Carolina;
5
Departments of Medicine and Pediatrics; University of North Carolina, Chapel Hill, North Carolina;
6
Institute for Environmental Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania.
7
Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania; mulugeta@mail.med.upenn.edu.

Abstract

Mutation of threonine for isoleucine at codon 73 (I73T) in the human surfactant protein C (hSP-C) gene (SFTPC) accounts for a significant portion of SFTPC mutations associated with interstitial lung disease (ILD). Cell lines stably expressing tagged primary translation product of SP-C isoforms were generated to test the hypothesis that deposition of hSP-C(I73T) within the endosomal system promotes disruption of a key cellular quality control pathway, macroautophagy. By fluorescence microscopy, wild-type hSP-C (hSP-C(WT)) colocalized with exogenously expressed human ATP binding cassette class A3 (hABCA3), an indicator of normal trafficking to lysosomal-related organelles. In contrast, hSP-C(I73T) was dissociated from hABCA3 but colocalized to the plasma membrane as well as the endosomal network. Cells expressing hSP-C(I73T) exhibited increases in size and number of cytosolic green fluorescent protein/microtubule-associated protein 1 light-chain 3 (LC3) vesicles, some of which colabeled with red fluorescent protein from the gene dsRed/hSP-C(I73T). By transmission electron microscopy, hSP-C(I73T) cells contained abnormally large autophagic vacuoles containing organellar and proteinaceous debris, which phenocopied ultrastructural changes in alveolar type 2 cells in a lung biopsy from a SFTPC I73T patient. Biochemically, hSP-C(I73T) cells exhibited increased expression of Atg8/LC3, SQSTM1/p62, and Rab7, consistent with a distal block in autophagic vacuole maturation, confirmed by flux studies using bafilomycin A1 and rapamycin. Functionally, hSP-C(I73T) cells showed an impaired degradative capacity for an aggregation-prone huntingtin-1 reporter substrate. The disruption of autophagy-dependent proteostasis was accompanied by increases in mitochondria biomass and parkin expression coupled with a decrease in mitochondrial membrane potential. We conclude that hSP-C(I73T) induces an acquired block in macroautophagy-dependent proteostasis and mitophagy, which could contribute to the increased vulnerability of the lung epithelia to second-hit injury as seen in ILD.

KEYWORDS:

alveolar epithelium; amphisome; autophagy; pulmonary fibrosis; surfactant protein

PMID:
25344067
PMCID:
PMC4281696
DOI:
10.1152/ajplung.00217.2014
[Indexed for MEDLINE]
Free PMC Article

Publication types, MeSH terms, Substances, Grant support

Publication types

MeSH terms

Substances

Grant support

Supplemental Content

Full text links

Icon for Atypon Icon for PubMed Central
Loading ...
Support Center